Polymyxin B (PB) is widely used as a last selection of infection therapy due to the emergence of multi-drug resistant bacteria. The commercial formulation of PB is a chemical mixture containing over 30 polymyxin B polypeptides. It was reported that there were variations in the composition of PB components in different products. The different components may not exhibit equivalent pharmacological activity and toxic propensity. Therefore, monitoring of all the main forms of polymyxin B is needed for accurate assessment of their pharmacokinetic properties and toxicity. Thus, we develop a simple, sensitive and selective LC/MS/MS method for the quantitation of four main forms of polymyxin B in human plasma including PB1, PB2, PB3 and Ile-PB1, which account for more than 95% of the polypeptides in commercial formulation.